Back to EveryPatent.com
| United States Patent |
5,055,485
|
|
Geacintov
, et al.
|
October 8, 1991
|
Inactivation of viruses in cell- and protein-containing compositions
using aryl diol epoxides
Abstract
A process of inactivating infectious viruses in a cell-containing or a
protein-containing composition containing such viruses, comprising
contacting such composition with an effective amount of at least one aryl
diol epoxide of the formula
##STR1##
in which X is an aromatic ring system having from 3 to 6 used rings,
for a sufficient period of time substantially to inactivate said viruses
without incurring substantial disruption or inactivation of cells or
without incurring substantial protein denaturation.
| Inventors:
|
Geacintov; Nicholas E. (New York, NY);
Valinsky; Jay E. (New York, NY);
Williams; Bolanle (Forest Hills, NY);
Horowitz; Bernard (New Rochelle, NY)
|
| Assignee:
|
New York Blood Center, Inc. (New York, NY)
|
| Appl. No.:
|
279179 |
| Filed:
|
December 2, 1988 |
| Current U.S. Class: |
514/449; 424/529; 424/530; 424/531; 424/583; 435/2; 514/2 |
| Intern'l Class: |
A61K 031/335 |
| Field of Search: |
435/1,2
514/449
|
References Cited
Other References
Yagi et al. J. Am. Ch. Soc. vol. 99(1977) pp. 1604-1611.
Wislocki et al.--Cancer Research vol. 36 Sep. 1976 pp. 3350-3357.
Wood et al.--Cancer Research vol. 36 (Sep. 1976) pp. 3358-3366.
|
Primary Examiner: Rosen; Sam
Attorney, Agent or Firm: Sprung Horn Kramer & Woods
Claims
What is claimed is:
1. A process of inactivating infectious viruses in a cell-containing or a
protein-containing composition containing such viruses, comprising
contacting such composition with an effective amount of at least one aryl
diol epoxide of the formula
##STR3##
in which X is an aromatic ring system having from 3 to 6 fused rings,
for a sufficient period of time substantially to inactivate said viruses
without incurring substantial disruption or inactivation of cells or
without incurring substantial protein denaturation.
2. A process according to claim 1, wherein X includes at least one side
group selected from the group consisting of primary amines, substituted
amines of the formula --NR.sub.2 where R is an aliphatic alkyl chain of 1
to 3 carbon atoms, sulfonic acids and hydroxyalkyl derivatives having 1 to
3 carbon atoms.
3. A process according to claim 2, wherein the aryl diol epoxide is
selected from the group consisting of trans-7,8-dihydroxy [anti] 9,10,
epoxy-7,8,9,10 tetrahydro benzo[a]pyrene, methylcholanthrene diol epoxides
and benzophenanthrene diol epoxides.
4. A process according to claim 2, wherein the contacting is conducted at a
temperature of 4.degree. to 37.degree. C.
5. A process according to claim 2, wherein the contacting is conducted at a
temperature of 22.degree. C. to 30.degree. C.
6. A process according to claim 2, wherein the contacting is conducted for
a time of 1 to 18 hours.
7. A process according to claim 2, wherein the contacting is conducted for
a time of 2 to 6 hours.
8. A process according to claim 2, wherein the contacting is conducted at a
pH of 6.8 to 7.5.
9. A process according to claim 2, wherein the aryl diol epoxide is at a
concentration of 1 to 500 .mu.M.
10. A process according to claim 2, wherein the concentration is 1 to 10
.mu.M.
11. A process according to claim 2, wherein said protein-containing
composition is selected from the group consisting of whole mammalian
blood, blood cell proteins, milk, saliva, blood plasma, a plasma
concentrate, a precipitate from any fractionation of said plasma, a
supernatant from any fractionation of said plasma, a serum, a
cryoprecipitate, a cryosupernatant, a cell lysate, placental extracts and
products of fermentation.
12. A process according to claim 2, wherein said cell-containing
composition includes one or more components selected from the group
consisting of red blood cell concentrates, platelet concentrates,
leukocyte concentrates, semen, ascitic fluid, hybridoma cell lines and
whole mammalian blood.
13. A process according to claim 2, wherein said composition is the product
of a non-blood normal or cancerous cell or the product of gene splicing.
14. A process according to claim 2, further comprising heat treating the
composition.
15. A process according to claim 2, further comprising contacting the
composition with solvents, detergents or solvents and detergents.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to producing virus-free biological mixtures such
that valuble biological components, e.g., cells and proteins, retain their
structure and function. More especially, this invention relates to the
inactivation of viruses, e.g., hepatitis viruses, human immunodeficiency
viurus (HIV), or other virues in human blood, blood cellular components,
blood plasma and blood plasma fractions such that each remains suitable
for therapeutic use. In particular, this invention relates to producing
cellular blood products (e.g., whole blood, red cell concentrates,
platelet concentrates and leukoiyte concentrates) and non cellular blood
products (e.g., whole plasma, antihemophilic factor immune globulin,
fibrinogen) which are rendered substantially free of infectious hepatitis
B virus, non-A, non-B hepatitis virus, or other viral infectivity by
treatment with aryl diol epoxides.
2. Background Information
Transmission of viral diseases (e.g., hepatitis B, acquired
immunodeficiency syndrome, cytomegalovirus infections) through blood
transfusion is a significant problem in transfusion medicine. While donor
selection criteria and screening of donor blood for viral markers helps
reduce the transmission of viruses to recipients, screening methods are
incomplete, and it is desirable to inactivate viruses contained in donor
blood without altering the structure and function of its valuable
consitituents, e.g., red blood cells, platelets, leukocytes, and plasma
proteins. Similarly, other biological mixtures, e.g., hybridoma cell
lines, milk and sperm, can contain infections virus and it would be
advantageous to inactivate said virus(es) while retaining the valuable
consitutents of these mixtures.
Methods typically used for the inactivation viruses, such as those useful
in the preparation of viral vaccines, generally destroy the function and
structure of cells and proteins. For instance, in the preparation of a
hepatitis B virus vaccine, it is common practice to heat the prearation at
temperatures in excess of 80.degree. C. and to treat with formaldehyde.
These treatments not only inactivate viral infectivity, but also
irreversibly damage blood cells and proteins, rendering them unsuitable
for transfusion.
Recently, both physical and chemical methods have been developed which
inactivate viruses contained in blood, while retaining blood protein
structure and function. Protein solutions heated either following
lyophilization or in solution in the presence of high concentrates of
sugars and/or amino acids have been shown to have greatly reduced viral
infectivity, while retaining functional activity of many proteins.
Chemical methods in use for the preparation of protein mixtures include
beta-propiolactone and the use of solvent/detergent mixtures, especially
tri(n-butyl)-phosphate. However, application of these methods to cells
generally results in disruption and inactivation.
As a result of the foregoing, viral inactivation methods are not commonly
applied to the preparation of whole blood and blood cell components
derived therefrom. Rather, viral safety relies solely on donor selection
and donor blood screening; methods known to be useful, but insufficient.
Thus, the recipients of these products must accept the risk that they may
be contaminated with hepatitis viruses, HIV, cytomegalovirus or other
infectious viruses. As a result, these recipients may suffer liver damage
or damage to other organ systems, illness, incapacitation, and
occassionally death.
Other methods for inactivation of viruses in cellular products include
ultraviolet light, gamma-irradiation, or the use of beta-propiolactone.
Each method can be characterized as being non-specific, modifying nucleic
acid, other cell structures and proteins alike. Thus, for example, the use
of ultraviolet light has been shown to inactivate viruses in a platelet
concentrate, however, severe platelet damage resulted from higher
intensitites. Beta-propiolactone reacts with nucleic acid and protein with
similar rate constants; thus, while viruses can be activated, more than
half of the factor VIII content of plasma is lost.
Yet another problem is that some of the viruses contaminating blood or
other biological fluids are contained within the cell, either as a fully
formed virus or in the form of free viral nucleic acid integrated into the
host genome. For instance, the human immunodeficiency virus is contained
within leukocytes. It is a special concern to be able to inactivate both
cell-free and cell-contained forms of virus, while retaining the structure
and function of cells not containing virus. Fortunately, not all viruses
are contained within the cells of interest and the functionality of some
cells, e.g., red blood cells or platelets, do not require cell division
following transfusion.
It is to be understood that the problems of inactivation of the viruses in
valuable biological mixtures are distinct from the problems of
inactivation of the viruses themselves due to the copresence of the
desirable proteinaceous components of the plasma. Thus, while it is known
how to inactivate the hepatitis B virus by using crosslinking agents, for
example, glutaraldehyde, nucleic acid reacting chemicals, for example,
formaldehyde, or oxidizing agents, for example chlorox, etc., it has been
believed that these methods are not suitable for the inactivation of the
virus in blood due to the observation that most of these activating
agents, e.g., glutaraldehyde, sodium hypochlorite or formaldehyde,
denatured the valuable proteinaceous components of the plasma.
Problems may also exist in deriving valuable proteins from non-blood
sources. These sources include, but are not limited to, mammalian milk,
ascitic fluid, serum, saliva, placental extracts, tissue culture cell
lines and their extracts, including transformed cells, and products of
fermentation. For instance, the human lymphoblastoid cells have been
isolated which produce alpha-interferon. However, the cell line in
commercial use today contains Epstein-Barr virus genes. It has been a
major concern that the use of interferon produced by these cells would
transmit viral infection or induce viral caused cancerous growth.
The present application concerns the action of aryl diol epoxides to
inactive viruses and simultaneously to retain labile protein activity.
Aryl diol epoxides are known to form adducts with DNA in vitro and in vivo
(H. B. Gamper et al, (1980), Proc. Nat. Acad. Sci. (USA), 77:2000).
Several studies have indicated anti-viral effects of these agents (M. L.
Lockhart et al, (1986), Chemico-Biological Interact., 58: 217; G. T. Chang
et al, (1981), Biochem. Biophys Res. Comm., 100:1337; and G. T. Bowden et
al, (1986), Chemico-Biological Interact., 58:333). However, heretofore
there was no indication that these compounds have been used as antiviral
agents specifically in the context of the sterilization of cellular
components of blood.
The present invention is directed to achieving three goals, namely, (1) a
safe, (2) viral inactivated protein-containing composition, (3) without
incurring substantial protein denaturation. As shown above, these three
goals are not necessarily compatible since, for example, glutaraldehyde
inactivates viral infectivity, but fixes cells and substances such as
beta-propiolactone inactivate viruses, but also substantially denaturate
valuable plasma proteins, for example, factor VIII.
It, therefore, became desirable to provide a virus inactivation process for
obtaining cell- or protein-containing compositions which does not
substantially inactivate cells or denature the valuable protein components
therein. More especially, it is desirable to provide blood cell and blood
protein-containing compositions in which substantially all of the
hepatitis viruses and other viruses present are inactivated, but which
retains at least 60% and preferably 80% intact cells, (e.g., red blood
cells) and active protein (e.g., immunoglobulin).
It is a further object of the present invention to provide products from
other biological fluids, from cancer or normal cells or from fermentation
processes following gene insertion which are substantially free of
infectious virus.
SUMMARY OF THE INVENTION
It has now been discovered that while most of the viral inactivating agents
disrupt red blood cells and denature factor VIII and other valuable blood
plasma proteins, that not all viral inactivating agents have such effect.
It has been discovered that when a cell- or a protein-containing
composition such as whole blood, red cell concentrates, platelet
concentrates, leukocyte concentrates, blood cell proteins, a blood plasma
protein fraction, milk, serum, semen, mammalian milk, placental extracts,
products of fermentation, ascities fluid, a non-blood product produced
from normal or cancerous cells (e.g., via recombinant DNA technology) is
contacted for a sufficient period of time with an effective amount of an
aryl diol epoxide that viruses such as the hepatitis viruses present in
the composition are virtually entirely inactivated, without substantial
denaturation of proteins contained therein. By contacting blood or a
concentrate thereof or a fraction thereof with an aryl diol epoxide,
hepatitis viruses can be substantially inactivated, e.g., to an
inactivation of greater than 4 logs, while realizing a yield of intact
cells as compared to total cells prior to virus inactivation and a yield
of protein activity to total protein of at least 80%.
Aryl epoxide hydrolyzes to non-toxic derivatives thereof during and
following the period critical to virus inactivation.
By such inventive procedures there is provided a process providing cell- or
protein-containing composition such as mammalian whole blood, red cell
concentrates, platelet concentrates, leukocyte concentrates, milk, semen
or blood protein derivatives, having greatly reduced or vitually no
hepatitis viruses or other viruses.
By the inactivation procedure of the invention, most if not virtually all
of the viruses contained therein would be inactivated. A method for
determining infectivity levels by in vivo chimpanzees is discussed by
Prince, A. M., Stephen, W., Brotman, B. and van den Ende, M. C.,
"Evaluation of the Effect of Beta-propiolactone/Ultraviolet Irradiation
(BPL/UV) Treatment of Source Plasma on Hepatitis Transmission by Factor IX
Complex in Chimpanzees", Thrombosis and Haemostasis, 44: 138-142, (1980).
According to the invention, inactivation of virus is obtained to the extent
of at least "4 logs", i.e., virus in a serum is totally inactivated to the
extent determined by infectivity studies where that virus is present in
the untreated serum in such a concentration that even after dilution to
10.sup.4, viral activity can be measured.
DETAILED DESCRIPTION OF THE INVENTION
Blood is made up of solids (cells, i.e., erythrocytes, leucocytes, and
platelets) and liquid (plasma). The cells are transfused in the treatment
of anemia, clotting disorders, infections, etc. In addition, the cells
contain potentially valuable substances such as hemoglobin, and they can
be induced to make other potentially valuable substances such as
intereron, growth factors, and other biological response modifiers. The
plasma is composed mainly of water, salts, lipids and proteins. The
proteins are divided into groups called fibrinogens, serum globulins and
serum albumins. Typical antibodies (immune globulins) found in human blood
plasma include those directed against infectious hepatitis, influenza H,
etc.
Blood transfusions are used to treat anemia resulting from disease or
hemorrhage, shock resulting from loss of plasma proteins or loss of
circulating volume, diseases where an adequate level of plasma protein is
not maintained, for example, hemophilia, and to bestow passive
immunization.
With certain diseases one or several of the components of blood may be
lacking. Thus the administration of the proper fraction will suffice, and
the other components will not be "wasted" on the patient; the other
fractions can be used for another patient. The separation of blood into
components and their subsequent fractionation allows the proteins to be
concentrated, thus permitting concentrates to be treated. Of great
importance, too, is the fact that the plasma fractions can be stored for
much longer periods than whole blood and they can be distributed in the
liquid, the frozen, or the dried state.
Cell types found in human blood include red blood cells, platelets and
several types of leukocytes. Methods for the preparation of cell
concentrates useful in transfusion can be found in Kirk Othmer's
Encyclopedia of Chemical Technology, Third Edition, Interscience
Publishers, Volume 4, pp 25-37, the entire contents of which are
incorporated by reference herein.
Proteins found in human plasma include prealbumin, retinol-binding protein,
albumin, alpha-globulins, beta-globulins, gamma-globulins (immune serum
globulins), the coagulation proteins (antithrombin III, prothrombin,
plasminogen, antihemophilic factor-factor VIII, fibrin-stabilizing
factor-factor XIII and fibrinogen), immunoglobins (immunoglobulins G, A,
M, D, and E), and the complement components. There are currently more than
100 plasma proteins that have been described. A comprehensive listing of
such plasma proteins can be found in "The Plasma Proteins", ed. Putnam, F.
W., Academic Press, New York (1975).
Blood plasma fractionation generally involves the use of organic solvents
such as ethanol, ether and polyethylene glycol at low temperatures and at
controlled pH values to effect precipitation of a particular fraction
containing one or more plasma proteins. The resultant supernatant can
itself then be precipitated and so on until the desired degree of
fractionation is attained. More recently, separations are based on
chromatographic processes. An excellent survey of blood fractionation
appears in Kirk-Othmer's Encylopedia of Chemical Technology, Third
Edition, Interscience Publishers, Volume 4, pages 37 to 62, the entire
contents of which are incorporated by reference herein.
Proteins found in the blood cell fraction include hemoglobin, fibronectin,
fibrinogen, enzymes of carbohydrate and protein metabolism, etc. In
addition, the synthesis of other proteins can be induced, such as
interferons and growth factors.
A comprehensive list of inducible leukocyte proteins can be found in
Stanley Cohen, Edgar Pick, J. J. Oppenheim, "Biology of the Lymphokines",
Academic Press, New York, (1979).
The present invention is directed to contacting one or more aryl diol
epoxides with a blood cell or blood protein-containing composition such as
whole mammalian blood, blood cells thereof (e.g., red blood cells, white
blood cells, platelets), blood cell proteins, blood plasma thereof, single
donor and apheresis derived platelets, single donor plasma, precipitates
from any fractionation of such plasma, supernatants from any fractionation
of such plasma, cryoprecipitate, cryosupernatant or any portion or
derivatives of the above that contain blood proteins such as, for example,
prothrombin complex (factors II, VII, IX and X) and cryoprecipitate
(factors I and VIII).
The present invention is also concerned with contacting one or more aryl
diol epoxides with a serum containing one or more blood proteins.
Furthermore, the present invention is directed to contacting one or more
aryl diol epoxides with a blood protein-containing fraction containing at
least one blood protein such as the following: factor II, factor VII,
factor VIII, factor IX, factor X, fibrinogen and IgM.
Additionally, the present invention concerns contacting any other cell- or
protein-containing composition such as a cell lysate or proteins induced
in blood cells, milk, serum, semen, ascities fluid or tissue culture
fluid, with one or more aryl diol epoxides.
Aryl diol epoxides for use in the present invention can be represented by
the following formulas:
##STR2##
The number of rings in the aromatic ring system in the above formulas may
generally range from 3 to 6.
Polycyclic aryl diol epoxides, typified by benzo (a) pyrene diol epoxide,
are only sparingly soluble in aqueous media (1-10 .mu.M). Because of their
limited solubility in aqueous solutions, and their generally hydrophobic
nature, these compounds can form non-covalent or physical complexes with
DNA, proteins and biological membranes. Because they are hydrophobic,
these compounds can also traverse cell membranes and subsequently
intercalate into cellular DNA. This non-covalent binding to DNA results in
the formation of DNA adducts which render the nucleic acid inactive. This
same principle applies to intercalation of aryl diol epoxides into viral
nucelic acids. The affinity of diol epoxides for DNA is such that
extensive covalent bonding to the nucleic acid fraction occurs even in the
presence of proteins to which aryl epoxides also bind noncovalently and
covalently. A second important property of these molecules is that they
are readily hydrolyzed in aqueous media (e.g., the half-life of
benzopyrene (a) diol epoxide in Iscove's medium containing 10% fetal
bovine serum is approximately 30 minutes). Thus, while the native
compounds may be potentially carcinogenic, the hydrolysis products are
not.
Non-limiting examples of polycyclic aryl diol epoxides for use in the
present invention include trans-7,8-dihydroxy [anti] 9,10
epoxy-7,8,9,10-tetrahydro benzo[a]pyrene ("BAPDE") and analogs of BAPDE,
such as, 3-methylcholanthrene diol epoxides and benzophenanthrene diol
epoxides.
The physical binding of aryl diol epoxides to hydrophobic regions of
proteins and biological membranes may result in an undesirable competitive
reaction which may limit the extent to which these molecules react with
DNA. By modifying the chemical structures of BAPDE and related molecules,
their physical binding to hydrophobic cell components may be minimized,
without significant affecting their chemical reactivities with DNA. In
this manner, the inactivation of viruses in the presence of blood cells or
other biological fluids, can be maximized.
The modification involve the synthesis of BAPDE and related compounds with
side groups located on the polycyclic aromatic ring systems which will
increase their water solubility, but will not markedly affect interactions
with DNA. These side groups include, but are not limited to, primary
amines (--NH.sub.2), substituted amines (--NR.sub.2, where R is an inert
aliphatic alkyl chain of 1 to 3 carbon atoms), sulfonic acids (--SO.sub.3
--) or hydroxyalkyl derivatives having 1 to 3 carbon atoms. An example of
this principle is seen in proflavin and acridine orange, two well known
polynuclear aromatic molecules with --NH.sub.2 and --N(CH.sub.3).sub.2
side chains which bind extremely well to DNA but which are also water
soluble.
The positions of substituents which impart enhanced solubility on BAPDE and
related molecules on the aromatic rings can be selected to limit the
effects of these side groups on binding to DNA. For example, substitution
at the 2,3,4 or 5 positions of BAPDE should have a minimal effect on the
reactivity with DNA, while at the same time significantly enhancing the
water solubility of the molecule.
Non-limiting examples of viruses than can be inactivated by the present
invention include vesicular stomatitis virus (VSV), Moloney sarcoma virus,
Sindbis virus, human immunodeficiency viruses HIV-1; HIV-2), human T-cell
lymphotrophic virus-I (HTLV-I), hepatitis B virus, non B, non A hepatitis
virus (NANB), cytomegalovirus, Epstein Barr viruses, lactic dehydrogenase
viruses, herpes group viruses, rhabdoviruses, leukoviruses, myxoviruses,
alphaviruses, Arboviruses (group B), paramyxoviruses, arenaviruses and
coronaviruses.
Assays of the effects of polycyclic aryl diol epoxides on the inactivation
of these viruses may require the use of cell lines (e.g., A549), enzyme
assays (reverse transcriptase, blood clotting times), assessment of
functional, biochemical and structural integrity of cells (e.g.,
metabolite assays, platelet aggregometry, polyacrylamide gel
electrophoresis) or the application of molecular biological techniques
(e.g., infusion of animals with process cellular blood components or blood
derivatives to assess infectivity, red cell survival studies, platelet
survival studies).
The process of the present invention is preferably conducted at 4.degree.
to 37.degree. C., and most preferably at 22.degree. to 30.degree. C. for 1
to 18 hours and preferably for 2 to 6 hours.
Preferably the concentration of the aryl diol epoxide is 1 to 500 .mu.M,
and most preferably is 1 to 10 .mu.M.
The treatment according to the present invention is normally carried out at
atmospheric pressure, although subatomospheric pressures can also be
employed.
Preferred treatment conditions including following:
Reagents used: BAPDE (1-10 .mu.M) from 1-3 mM stock in tetrahydrofuran,
triethanolamine or other aprotic solvents.
Temperature: 24.degree. C.
pH: 7.2 (6.8-7.5)
Time: 4 hours (2-6 hours)
Cell concentration:
(a) packed red cells (approximately 10.sup.10 per ml)
(b) platelet concentrate (greater than 10.sup.10 per ml)
(c) whole blood
Protein concentration:
(a) plasma (approximately 60 g/l)
(b) AHF
(c) fibronectin
After treatment of cell preparation the hydrolysis products of the
inactivating agent will not typically be removed. In some instances,
however, (e.g., during the washing of red cells as part of
freezing/thawing procedures or during the preparation of purified protein
products from plasma or other sources), the hydrolysis products may be
removed.
The process of the invention can be combined with still other modes of
inactivating viruses. For instance, in the case of platelet concentrates,
treatment with aryl diol epoxides can be supplemented by treatment with
ultraviolet irradiation. Such treatment, effected with a pulsed
ultraviolet (UV) laser beam, could render certain viruses inactive,
without significantly impairing platelet function.
In another instance, virus inactivation in protein solutions treated with
aryl diol epoxides could be supplemented with treatment with tri-N-butyl
phosphate and detergents such as Tween 80 or cholic acid. Such
supplemental treatments inactivate viruses, but permit retention of at
least 80% of the activity of proteins such as factor VIII.
The present invention describes inactivating viruses, while simultaneously
retaining labile blood cell functions and structural features, and in the
case of proteins derived from blood or other sources, while retaining
enzymatic, binding or other activities.
Functional activities of red cells are ascertained by measurements of
metabolite levels, enzymatic activities, electrolyte levels and oxygen
carring capacity. Structural integrity of red cells is assessed by
measurements of osmotic fragility, survival in vivo following
radiolabeling with chromium-51, antigenicity and by evaluation of
modification of cell surface proteins.
Functional activities of platelets are determined by their ability to
aggregate in the presence of certain biological agents, morphology and
rate of fall in pH in the preparation. Structural integrity of platelets
is assessed by in vivo survival following radiolabeling with indium-111
and identification of the presence of specific platelet antigens.
The activity of proteins which are enzymes is determined by measuring their
enzymatic activity (e.g., factor IX).
The activity of binding proteins can be measured by determining their
kinetics and affinity of binding to their natural ligands.
Lymphokine activity can be measured biologically in cell systems, typically
by assaying their biological activity in cell cultures.
Protein activity generally is determined by the known and standard modes
for determining the activity of the protein or type of protein involved.
In order to more fully illustrate the nature of the invention and the
manner of practicing the same, the following non-limiting examples are
presented:
EXAMPLES
Example 1
Vesicular stomatitis virus (VSV) was incubated, at temperatures between
4.degree. and 30.degree. C., in Iscove's medium containing 10% fetal
bovine serum, in the presence of trans-7,8-dihydroxy [anti] 9,10, epoxy-
6,8,9,10 tetrahydro benzo[a]pyrene (BAPDE) at a concentration of 1-10
.mu.M for 0-6 hours. Virus titer was ascertained following incubation of
the supernatant suspension with A549 cells; virus kill was inversely
proportional to the appearance of cytopathology in the cell cultures.
BAPDE effectively inactivated in excess of 5 logs of virus under these
conditions compared to controls in which BAPDE was omitted. Neither BAPDE
nor the solvent in which it was dissolved (e.g., tetrahydroturan) were
toxic to A549 cells under these conditions.
Example 2
In a second series of experiments, VSV was exposed to BAPDE under the same
experimental conditions in Example 1, except that washed human red blood
cells were added at concentrations between 10.sup.4 and 10.sup.9 cells/ml.
VSV inactivation was reduced in the presence of red cells, but the virus
titer was reduced at least 3 logs relative to controls in which BAPDE was
omitted.
Red cell structural and functional integrity was not compromised under the
experimental conditions described. Measurements of osmotic fragility,
hemoglobin content, ATP levels and 2,3 diphosphoglycerate levels were all
within the normal range.
Example 3
The preceding examples illustrate the BAPDE can effectively kill virus
which is free in suspension in the presence of red cells. An equally
important application is the inactivation of viruses which have become
cell-associated or incorporated into target cells.
VSV (10.sup.6 infectious units) was incubated with A549 cells for 6 hours
at 37.degree. C. During this period, the virus was taken up by the cells
and entered a replicative cycle. The cells were washed extensively to
remove non-cell associate virus, removed from the tissue culture plates
and suspended in Iscove's medium containing 4% fetal bovine serum. The
cells were exposed to BAPDE at 3 .mu.M. The preparation was incubated at
24.degree. C. for 0-6 hours and virus titer assessed by assay on fresh
A549 cells. BAPDE effected greater than 5.5 logs of virus kill under these
conditions compared to control preparations from which BAPDE was omitted.
Example 4
Human plasma was incubated with BAPDE at a concentration of 10 .mu.m for 6
hours at 24.degree. C. Prior to incubation, VSV was added to the plasma to
serve as a marker of virus kill. The functional activity of selected
plasma proteins and the infectivity of VSV was assessed prior to and
following incubation with BAPDE. The results in the accompanying Table
indicate virus inactivation to the extent of greater than 4.5 log.sub.10
without significant loss of protein functional activity.
TABLE I
______________________________________
NO FOLLOWING
TREAT- TREATMENT
MENT with BAPDE CHANGE
______________________________________
VSV Infectivity
4.1 <-0.5 log.sub.10 Kill
(log ID.sub.50) >4.6
Recovery (%)
Anti-HBsAg (titer)
0.50 0.55 110%
Fibrinogen clottable
clottable 100%
clottability
AHF (units/ml)
0.70 0.55 79%
Factor IX 0.9 0.9 100%
(units/ml)
Haptoglobin 175 170 97%
(mg/dl)
______________________________________
It will be appreciated that the instant specification and claims are set
forth by way of illustration and not limitation, and that various
modifications and changes may be made without departing from the spirit
and scope of the present invention.
Top